1. Field of the Invention
The present invention relates to chemical compositions of preenrichment broth and methods which allow the simultaneous sampling of food products for the human bacterial enteropathogens Salmonella and Listeria.
2. Description of the Prior Art:
Two to three million cases of Salmonella food poisoning are estimated to occur each year in the United States. Acute symptoms of this disorder include nausea, vomiting, diarrhea, cold chills, fever, and exhaustion. About 2,000-3,000 (0.1%) people die each year in the United States from Salmonella poisoning, with these victims usually being infants, the sick, and elderly. Seventeen hundred to 1800 cases of Listeria food poisoning are estimated to occur each year in the United States.
Acute symptoms include septicemic meningitis with mild flu-like symptoms reported in some cases. About 500 people (25 to 30%) people die each year in the United States from Listeria food poisoning, with the victims usually being the immunocompromised, pregnant women or neonates.
Poultry, red meat, seafood, eggs and any foods which contain these products or which come in contact with fecal material have the potential to carry the human bacterial enteropathogens Salmonella and Listeria. The challenge to a food microbiologist is to be able to recover small numbers of these select pathogens from food products which often contain large numbers of a variety of other bacteria. Recovery of sublethally injured Salmonella and Listeria is even more difficult. Heating, freezing, chemicals and other processing steps can sublethally injure or debilitate pathogenic bacteria in foods.
Traditional recover methods for foodborne Salmonella involve 5 basic steps:
1. Preenrichment--The initial step on which the food sample is enriched in a nonselective medium to restore injured Salmonella cells to a stable physiological condition. PA0 2. Selective enrichment--A step in which the sample is further enriched in growth-promoting medium containing selectively inhibitory reagents. This medium allows a continued increase of Salmonella while simultaneously restricting proliferation of most other bacteria. PA0 3. Selective plating--A step using solid selective media that restrict growth of bacteria other than Salmonella and provide visual recognition of pure, discrete colonies suspected to be Salmonella. PA0 4. Biochemical screening--An elimination of most organisms other than Salmonella that also provides a tentative generic identification of Salmonella cultures. PA0 5. Serotyping--A serological technique which provides a specific identification of cultures.
Listeria spp. have the ability to grow slowly at refrigerated temperatures. Historically, recovery methods for Listeria used a nonselective, nutrient, enrichment broth which when incubated at refrigeration temperatures for up to a month allowed the Listeria to grow while concurrently suppressing the growth of competitive microorganisms. The major disadvantage of this procedure is that it is very time consuming. Newer more timely procedures have been developed utilizing selected antibiotics, other chemical inhibitors, and elevated incubation temperatures. These procedures are quite similar to the 5 step procedure for recovery of Salmonella. However, unlike the Salmonella preenrichment step, the first stage of Listeria enrichment uses broth which contains antibiotics and chemicals and can interfere with the repair and growth of sublethally injured Listeria.
There are basic differences in Salmonella and Listeria which make simultaneous recovery of these two organisms seem incompatible. Salmonella are Gram negative bacteria while Listeria are Gram positive. Most chemical inhibitors or antibiotics which are active against Gram negative bacteria have little effect on Gram positive bacteria and vice versa.
Current procedures for recovery of Salmonella from food use either lactose broth or buffered peptone (BP) as preenrichment broths. Current procedures for recovery of Listeria from foods use either Listeria enrichment broth (LEB) or University of Vermont (UVM) broth as modified by the Food Safety Inspection Service of the USDA as preenrichment broths. Lactose and BP do not contain any chemical or antibiotic inhibitors. LEB and UVM contain both antibiotic and chemical inhibitors which favor the growth of Listeria while inhibiting the growth of competitive microorganisms which may be present in the foods. Sublethally injured Listeria may not repair and grow in the presence of these chemical inhibitors which have little effect on injured cells.
We found that in the presence of foods containing large numbers of extraneous microorganisms LEB did not contain sufficient buffers to prevent a rapid drop in the pH of the broth and injured cells could not be recovered. The primary disadvantage of the currently used first stage enrichment broths for Salmonella and Listeria are that the same broth cannot be used to simultaneously sample food and environmental samples for both of these pathogens and that the first stage enrichment for Listeria contains antibiotics and chemical inhibitors which can inhibit the recovery of sublethally injured Listeria.
It is estimated that in 1989 over 5 million Salmonella and 3 million Listeria analyses were run on food products in the United States. Many of these samples were analyzed for both Salmonella and Listeria. It can be seen that if samples could be analyzed simultaneously for Salmonella and Listeria there would be a tremendous saving in the media required, incubator space required, and food product needed for analyses. Therefor it is an object of the present invention to provide a preenrichment broth and method by which Salmonella and Listeria can be sampled simultaneously.